1. Field of the Invention
The present invention relates to modified sarcosine oxidases having decreased reactivity for N-ethylglycine compared with that of an unmodified enzyme, modified sarcosine oxidase genes, and methods for preparing the modified sarcosine oxidases. Sarcosine is also known as N-methylglycine an amino acid found in muscles and other tissues. N-methylglycine (sarcosine) is chemically similar to N-ethylglycine (a metabolic product of certain anesthetics). Decreasing the reactivity of sarcosine oxidases for N-ethylglycine is important for improving the sensitivity and specificity of assays measuring sarcosine (N-methylglycine), especially in biological samples where N-ethylglycine may be present. Sarcosine is measured in conventional assays for creatinine and creatine which are important clinical indicators.
2. Background Art
Sarcosine oxidases are enzymes with catalytic activity to hydrolyze sarcosines to produce glycine and formaldehyde, which can be used to measure the amount of creatinine in human serum or urine, or can be utilized as diagnostic agents for various diseases such as renal disease.
It has previously been known that sarcosine oxidases are produced by bacterial strains such as those of the Corynebacterium genus (e.g., see J. Biochem., 89, 599 (1981)), Bacillus genus (e.g., see JP Patent Publication (Unexamined Application) No. 54-52789), Cylindrocarbons genus (e.g., see JP Patent Publication (Unexamined Application) No. 56-92790), Pseudomonas genus (e.g., see JP Patent Publication (Unexamined Application) No. 60-43379), and Arthrobacters genus (e.g., see JP Patent Publication (Unexamined Application) No. 2-265478). The polynucleotide or protein sequences for the sarcosine oxidases described by these documents are hereby specifically incorporated by reference.
The inventors have isolated sarcosine oxidase genes from the Bacillus genus (e.g., see SEQ ID NO: 2 of JP Patent Publication (Unexamined Application) No. 5-115281) (SEQ ID NO: 2) and succeeded in causing the enzymes to be expressed in a large amount by genetic engineering techniques (e.g., see JP Patent Publication (Unexamined Application) No. 5-115281). The polynucleotide or protein sequences for sarcosine oxidases described by these documents are also specifically incorporated by reference.
While sarcosine oxidases act on sarcosines (N-methylglycines) sarcosine oxidases are also known to react to N-ethylglycine, which is a metabolic product of anesthetics such as lidocaine. Since reagents containing sarcosine oxidases used therein are affected by N-ethylglycine, it has been impossible to precisely measure creatinine or creatine using such reagents. For example, conventional sarcosine oxidases exhibit specificity and sensitivity problems for determining sarcosine levels in subjects receiving anesthetics which metabolize into N-ethylglycine, see e.g., Roberts et al., Clinical Chemistry 34: 2569–2572, 1988.
Sarcosine levels are enzymatically measured during conventional enzymatic assays of creatinine levels. The serum creatinine level is a conventional indicator of kidney function and a normal or usual value ranges from about 0.8 to 1.4 mg/dl. Conventional enzymatic assays for creatinine involve conversion of creatinine into creatine which is then converted into sarcosine and urea. Sarcosine oxidase subsequently converts sarcosine into glycine, formaldehyde and hydrogen peroxide. By determining the amount of sarcosine in a sample or by determining the reaction products of sarcosine after the action of sarcosine oxidase, the creatinine content in a biological sample may be determined.
Conventional assays for creatinine using sarcosine oxidase are well known and are also disclosed by U.S. Pat. Nos: 4,740,465, 4,845,029 and 4,950,609, which are incorporated by reference. Such assays are used to diagnose many conditions which are described by the MedLine Plus Medical Encyclopedia, “Creatinine-serum” (Feb. 11, 2004 update). For example, greater than normal levels may indicate acute tubular necrosis, dehydration, diabetic nephropathy, eclampsia (a condition of pregnancy that includes seizures), glomerulonephritis, muscular dystrophy, pre-eclampsia (pregnancy-induced hypertension), pyelonephritis, reduced renal blood flow (shock, congestive heart failure), renal failure, rhabdomyolysis or urinary tract obstruction. Lower than normal levels are associated with muscular dystrophy (late stage) and myasthenia gravis. Creatinine levels are also measured in conjunction with other diseases or disorders including acute nephritic syndrome Alport syndrome, atheroembolic renal disease, chronic renal failure, complicated UTI (pyelonephritis), Cushing's syndrome, dementia due to metabolic causes, dermatomyositis, digitalis toxicity, ectopic Cushing's syndrome, end-stage renal disease, epilepsy, generalized tonic-clonic seizure, Goodpasture's syndrome hemolytic-uremic syndrome (HUS), hepatorenal syndrome, IgM mesangial proliferative glomerulonephritis, interstitial nephritis, lupus nephritis, malignant hypertension (arteriolar nephrosclerosis), medullary cystic disease, membranoproliferative GN I, membranoproliferative GN II, Noninsulin-dependent diabetes mellitus (NIDDM), polymyositis (adult), prerenal azotemia, primary amyloid, rapidly progressive (crescentic) glomerulonephritis, secondary systemic amyloid, thrombotic thrombocytopenic purpura and Wilms' tumor.